ORIGINAL RESEARCH
published: 21 March 2017
doi: 10.3389/fmicb.2017.00477
Changes in Gene Transcription
Induced by Hydrogen Peroxide
Treatment of Verotoxin-Producing
Escherichia coli O157:H7 and
Non-O157 Serotypes on Romaine
Lettuce
Edited by:
Michael Gänzle,
University of Alberta, Canada
Reviewed by:
Alexander Gill,
Health Canada, Canada
Keith Warriner,
University of Guelph, Canada
*Correspondence:
Magdalena Kostrzynska
[email protected]
† Present
address:
Gui-Ying Mei,
School of Environmental Sciences,
University of Guelph, Guelph, ON,
Canada
Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 04 January 2017
Accepted: 08 March 2017
Published: 21 March 2017
Citation:
Mei G-Y, Tang J, Bach S and
Kostrzynska M (2017) Changes
in Gene Transcription Induced by
Hydrogen Peroxide Treatment
of Verotoxin-Producing Escherichia
coli O157:H7 and Non-O157
Serotypes on Romaine Lettuce.
Front. Microbiol. 8:477.
doi: 10.3389/fmicb.2017.00477
Gui-Ying Mei 1† , Joshua Tang 1 , Susan Bach 2 and Magdalena Kostrzynska 1*
1
Guelph Research and Development Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada, 2 Summerland
Research and Development Centre, Agriculture and Agri-Food Canada, Summerland, BC, Canada
Disease outbreaks of verotoxin-producing Escherichia coli (VTEC) O157:H7 and nonO157 serotypes associated with leafy green vegetables are becoming a growing
concern. A better understanding of the behavior of VTEC, particularly non-O157
serotypes, on lettuce under stress conditions is necessary for designing more effective
control strategies. Hydrogen peroxide (H2 O2 ) can be used as a sanitizer to reduce
the microbial load in leafy green vegetables, particularly in fresh produce destined for
the organic market. In this study, we tested the hypothesis that H2 O2 treatment of
contaminated lettuce affects in the same manner transcription of stress-associated and
virulence genes in VTEC strains representing O157 and non-O157 serotypes. Six VTEC
isolates representing serotypes O26:H11, O103:H2, O104:H4, O111:NM, O145:NM,
and O157:H7 were included in this study. The results indicate that 50 mM H2 O2 caused
a population reduction of 2.4–2.8 log10 (compared to non-treated control samples) in all
six VTEC strains present on romaine lettuce. Following the treatment, the transcription
of genes related to oxidative stress (oxyR and sodA), general stress (uspA and rpoS),
starvation (phoA), acid stress (gadA, gadB, and gadW), and virulence (stx1A, stx2A, and
fliC) were dramatically downregulated in all six VTEC serotypes (P ≤ 0.05) compared
to not treated control samples. Therefore, VTEC O157:H7 and non-O157 serotypes
on lettuce showed similar survival rates and gene transcription profiles in response
to 50 mM H2 O2 treatment. Thus, the results derived from this study provide a basic
understanding of the influence of H2 O2 treatment on the survival and virulence of VTEC
O157:H7 and non-O157 serotypes on lettuce.
Keywords: verotoxin-producing Escherichia coli, non-O157 serotypes, lettuce decontamination, hydrogen
peroxide treatment, gene transcription
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H2 O2 Treatment of Contaminated Lettuce
2015). In addition, using H2 O2 as a sanitizer on fresh produce
to reduce E. coli O157:H7 was investigated (Sapers et al., 2000;
Huang et al., 2012). However, the survival and gene transcription
of non-O157 VTEC serotypes under stress conditions on lettuce
remain largely unknown. In this study, the behaviors of O157:H7
and non-O157 VTEC serotypes in response to H2 O2 treatment
on lettuce were evaluated.
INTRODUCTION
Verotoxin-producing Escherichia coli (VTEC), also referred to
as Shiga toxin (Stx)-producing E. coli (STEC), often cause
life-threatening diseases, such as hemorrhagic colitis (HC)
and hemolytic uremic syndrome (HUS) (Croxen et al., 2013;
Karpman and Stahl, 2014; Karmali, 2017). Although VTEC
serotype O157:H7 is commonly identified in human diseases,
non-O157 serogroups have been increasingly associated with
serious outbreaks and were recently responsible for more than
50% of STEC illness in U.S. (Luna-Gierke et al., 2014; Parsons
et al., 2016; CDC, 2017). In 2015, the incidence of confirmed
VTEC non-O157 infections was 40% higher than in 2012–2014
(Huang et al., 2016). More than 70% of infections linked to nonO157 VTEC were caused by serotypes O26, O45, O103, O111,
O121, and O145 (termed the Top 6; Saito et al., 1998; Brooks et al.,
2005; Folster et al., 2011; Bradley et al., 2012; Brown et al., 2012).
In 2011, enteroaggregative E. coli O104:H4 caused the biggest
outbreak in Germany. This strain produces Stx2 and is one of the
most virulent strains of non-O157 VTEC (Buchholz et al., 2011;
Zangari et al., 2013; Karmali, 2017).
Fresh leafy green vegetables are an important part of
a healthy diet due to their richness in minerals, vitamins,
and phytochemicals. However, leafy green vegetables can
be contaminated by pathogenic bacteria such as VTEC
during growth, harvesting, and transportation leading to
subsequent illnesses and outbreaks (Wachtel et al., 2002;
Solomon et al., 2003; Islam et al., 2004; Delaquis et al., 2007).
A number of surveys have shown an increase in foodborne
outbreaks linked to contaminated leafy green vegetables (Harris
et al., 2003; Sivapalasingam et al., 2004; Rangel et al., 2005;
Lynch et al., 2009; Painter et al., 2013; Herman et al.,
2015). Although the contamination can be minimized by
preventing produce exposure to sources of pathogenic bacteria,
including contaminated water, soil, and animals, the occasional
contamination of leafy green vegetables on farms by VTEC
can still occur, which leads to contaminated produce entering
the processing lines (Olaimat and Holley, 2012). Therefore,
effectively reducing the contamination during processing is
crucial to ensure the safety of fresh leafy green vegetables.
Chlorine has been widely used as a sanitizer to reduce the
microbial load in fresh-cut vegetables (Beuchat et al., 1998).
However, chlorine may react to form potentially carcinogenic or
mutagenic products (Hurst, 1995). In addition, the by-products
formed when sodium hypochlorite (NaOCl—another sanitizer
commonly used in the fresh produce industry) reacts with
organic compounds, have been shown to increase the risk of
bladder cancer (Villanueva et al., 2004). Hydrogen peroxide
(H2 O2 ) is a potential alternative to chlorine treatment and breaks
down to ecologically friendly water and oxygen (Linley et al.,
2012). In addition, H2 O2 has been approved for use in organic
postharvest processing systems. As such, this sanitizer could be
used for decontamination of fresh produce destined for organic
market.
To date, several studies have been conducted on the gene
expression in pure culture of E. coli O157:H7 under oxidative
stress (Wang et al., 2009; Allen and Griffiths, 2012; Mei et al.,
Frontiers in Microbiology | www.frontiersin.org
MATERIALS AND METHODS
Bacterial Strains and Growth Conditions
Six VTEC strains were tested in this study: E. coli O157:H7
(EDL933) (ATCC 700972), O26:H11 (EC20070549),
O103:H2 (EC19970811), O104:H4 (NML#11-3088), O111:NM
(EC20070546), and O145:NM (EC19970355). E. coli EDL933 was
included in the present study because it is prototype O157:H7
strain. Serotypes O26, O103, O111, and O145 are predominant
non-O157 serotypes worldwide, therefore strains representing
these serotypes have been investigated in this study. In addition
VTEC O104:H4 isolated from German outbreak was selected,
because this strain is highly virulent and is linked to fresh produce
outbreak. All the strains except EDL933 were of human origin.
E. coli O157:H7 (EDL933) was isolated from raw hamburger
meat linked to an outbreak of HC. Further details regarding
these strains have been published previously (Mei et al., 2015).
E. coli O157:H7 (EDL933) possesses genes encoding Stx1 and
Stx2, intimin gene (eae) and flagellin gene (fliC). VTEC O26:H11
(EC20070549) and O103:H2 (EC19970811) do not have stx2
genes. VTEC strains O111:NM (EC20070546) and O145:NM
(EC19970355) do not have genes encoding Stx2 and flagellin.
Enteroaggregative E. coli strain O104:H4 (NML#113088) does
not possess genes encoding Stx1 and intimin. For simplicity,
VTEC strains will be referred to by their serotype as each strain
has its distinct serotype. All the strains were grown in Tryptic
soy broth (TSB) at 37◦ C with shaking (180 rpm) for 18 h.
Subsequently 0.5 mL of 18 h culture of each VTEC strain was
inoculated into 50 mL pre-warmed TSB and incubated for 3 h at
37◦ C with shaking (180 rpm). The bacterial cells in logarithmic
phase were collected, washed twice with sterile distilled water
and used to inoculate lettuce samples.
Hydrogen Peroxide Treatment of
Contaminated Lettuce
Romaine lettuce was purchased from a local grocery store in
Guelph. The outer leaves were removed and the remaining leaves
were cut into 4 × 10 cm slices. Twenty-five grams of the leaves
were weighed, placed into sterile polyethylene stomacher bags,
and inoculated with 5 mL of bacterial suspension (108 CFU/g).
The leaves were massaged for 2 min to distribute the bacterial
suspension evenly on the leaves. Subsequently, 195 mL of H2 O2
was added to the bag to achieve a final concentration of 50 mM.
The negative control sample contained lettuce leaves with 5 mL
of autoclaved distilled water and 195 mL H2 O2 . The non-treated
control sample contained 5 mL of bacterial suspension and
195 mL of autoclaved distilled water. The bags were incubated at
room temperature for 40 min. After incubation, 1 mL of culture
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H2 O2 Treatment of Contaminated Lettuce
was taken from each stomacher bag and serially diluted with
0.1% (w/v) buffered peptone water. Subsequently, 100 µL of each
dilution was spread on cefixime/tellurite—Sorbitol MacConkey
agar (CT-SMAC) plates in duplicate. The plates were incubated
for 18 h at 37◦ C, and the CFU/mL was calculated. Additionally,
4 mL samples from controls and each stomacher bag treated with
50 mM H2 O2 were collected for RNA extraction.
TABLE 1 | List of stress-associated and virulence genes tested for
differential gene transcription upon treatment of VTEC strains on lettuce
with hydrogen peroxide.
oxyR
DNA-binding transcriptional regulator OxyR
RNA Extraction and cDNA Synthesis
sodA
Manganese-containing superoxide dismutase
soxR
Redox-sensitive transcriptional activator of
oxidative stress regulon
Stress-associated and
virulence genes
Oxidative stress
Bacterial cells were concentrated by centrifugation before
RNA extraction. After taking samples from controls and each
stomacher bag treated with 50 mM H2 O2 , each 4 mL sample
was added to 8 mL (2 volume) of RNAprotect Bacteria
Reagent (Qiagen). The samples were centrifuged for 10 min
at 5000×g and supernatants were decanted prior to RNA
extraction. RNA was isolated using the RNeasy Mini Kit
(Qiagen, Mississauga, ON, Canada), following the manufacturer’s
instructions for Gram-negative bacteria. Contaminating genomic
DNA was removed from each RNA preparation using the
Turbo DNA-freeTM kit (Ambion, Cambridge, UK), according
to the manufacturer’s instructions for DNase treatments. Total
RNA concentration was determined using a Thermo Scientific
Nanodrop 2000 (ON, Canada). Subsequently, 0.3 µg of RNA was
reverse-transcribed using SuperScript III (Invitrogen, Carlsbad,
CA, USA) according to the manufacturer’s instructions. A no
reverse transcriptase control (No RT) was included for each
DNase-treated RNA sample.
General stress
uspA
Universal stress global response regulator
rpoS
Regulator of the general stress response (σS )
Starvation
phoA
Alkaline phosphatase
dps
DNA-binding protein from starved cells
Cold shock
cspA
Cold shock protein A
cspC
Cold shock protein C
cspE
Cold shock protein E
UV
mutS
DNA mismatch repair protein—Mutator S
Acid resistance
gadA
Glutamate decarboxylase A
gadB
Glutamate decarboxylase B
gadW
DNA-binding transcriptional activator GadW
Intimin
eae
Real-Time PCR
Intimin (adherence protein)
Toxin
All stress-associated and virulence genes tested in this study are
described in Table 1. Transcription of key stress and virulence
genes (oxyR, sodA, soxR, uspA, rpoS, phoA, dps, cspA, cspC, cspE,
gadA, gadB, gadW, mutS as well as eae, stx1A, stx2A, and fliC)
was evaluated using a comparative real-time PCR. Each 25-µL
reaction contained 1 µL of reverse-transcribed cDNA, 12.5 µL
of Power SYBR Green PCR Master Mix, 0.25 U AmpErase
Uracil N-Glycosylase (UNG; Applied Biosystems), 2.0 µM of
each primer and 6.25 µL nuclease-free water. All primers used
in the study were previously described (Mei et al., 2015).
Amplification and detection were carried out on a MX3500
Multiplex Quantitative PCR System (Stratagene, La Jolla, CA,
USA) with an initial temperature of 50◦ C for 2 min. Following
denaturation at 95◦ C for 10 min, reactions were cycled 40 times
as follows: amplification at 95◦ C for 15 s, annealing at 60◦ C for
30 s, extension at 72◦ C for 30 s. Subsequent melt curve analysis
involved heating the products to 95◦ C for 1 min, followed by
cooling to 55◦ C for 30 s, and heating to 95◦ C while monitoring
fluorescence. No template control and No RT were included
for each assay and no Ct values were obtained for all negative
controls after 40 cycles of PCR (data not shown). Several wellknown candidate reference genes including 16S rRNA, tufA/B,
mdh, pyrC, gatB, recA, serC, frr, rpsU, udp, mdoG, rpoA, and
arcA were tested for expression stability as previously described
(Mei et al., 2015). Only 16S rRNA gene expression was stable
in all VTEC strains on lettuce under experimental conditions.
Therefore, 16S rRNA was used as reference gene in this study to
normalize the data. Serial dilutions of the cDNA template were
R
Protein encoded/function
stx1A
Shiga-like toxin 1 A subunit
stx2A
Shiga-like toxin 2 A subunit
Motility
fliC
Flagellin
examined with 16S rRNA primers. Using 50-fold dilutions the Ct
value was around 12, and with other primers for different target
genes all the Ct values were between 20 and 35. Therefore, 50-fold
dilutions of cDNA samples were used in the experiment.
R
R
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Statistical Analysis
The effect of H2 O2 treatment on survival and gene transcription
of VTEC strains present on lettuce was investigated by at least
three independent experiments. Each biological sample was run
in duplicate on real-time RT PCR. Relative mRNA levels were
determined according to the method described by Hellemans
et al. (2007). Gene transcription data were analyzed using
Student’s t-test. Data on reduction of VTEC populations on
lettuce following exposure to H2 O2 were analyzed using one-way
ANOVA.
RESULTS
Treatment of contaminated lettuce with 50 mM H2 O2 for 40 min
reduced the populations of all VTEC strains tested in this study
by 2.4–2.8 log10 (Table 2). The differences in sensitivity to H2 O2
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H2 O2 Treatment of Contaminated Lettuce
gadW) and virulence (stx1A), were significantly (P ≤ 0.05)
downregulated. Whereas, genes for cold shock (cspC) and MMR
(mutS) were significantly (P ≤ 0.05) upregulated (Figure 4).
In E. coli O26:H11 present on lettuce, 9 out of 17 genes,
including genes associated with oxidative damage (oxyR and
sodA), general stress (uspA and rpoS), starvation (phoA), acid
resistance (gadA, gadB, and gadW), and virulence (eae, stx1A, and
fliC), were significantly (P ≤ 0.05) downregulated. Three genes,
dps (starvation-related), cspC and cspE (cold shock-related), were
significantly (P ≤ 0.05) upregulated. In addition, genes for cold
shock (cspA) and DNA damage repair (mutS) were upregulated
(Figure 5).
In E. coli O103:H2 on lettuce, genes related to oxidative
stress (oxyR and sodA), general stress (rpoS), starvation (phoA),
acid resistance (gadW), and virulence (stx1A and fliC) were
significantly (P ≤ 0.05) downregulated. In addition, two genes
associated with acid resistance—gadA and gadB, as well as uspA
gene were downregulated. Interestingly, soxR and cspC, were
significantly (P ≤ 0.05) upregulated. Genes encoding cold shock
protein (cspA and cspE), and intimin (eae) were also upregulated
(Figure 6).
Overall, most of the genes associated with stress and virulence
were downregulated in O157:H7 and non-O157 serotypes on
lettuce treated with 50 mM H2 O2 . Only three genes, associated
with cold shock (cspC and cspE) and MMR (mutS), were
upregulated in all VTEC strains tested (Figures 1–6).
TABLE 2 | Effect of H2 O2 treatment on populations of six VTEC strains on
lettuce.
E. coli strain
Mean log reduction ± SE
O157:H7 (EDL933)
2.42 ± 0.15
O26:H11 (EC20070549)
2.73 ± 0.25
O103:H2 ((EC19970811)
2.81 ± 0.15
O104:H4 (NML#11-3088)
2.53 ± 0.18
O111:NM (EC20070546)
2.82 ± 0.25
O145:NM (EC19970355)
2.52 ± 0.26
Data are expressed as mean log reduction ± SE.
treatment on lettuce between VTEC O157:H7 and non-O157
serotypes were not statistically significant (P > 0.05).
In addition, the influence of H2 O2 treatment on stress and
virulence gene transcription of six VTEC strains on romaine
lettuce was investigated. This study focused on well-known
virulence genes including genes encoding intimin (eae), Stx
genes stx1A and stx2A and flagellin genes (fliC) (Figures 1–6).
Transcription of key stress-associated genes such as genes
involved in response to oxidative damage (oxyR, sodA, and soxR),
general stress (uspA and rpoS), and starvation (phoA and dps)
was investigated. The study also focused on the effects of H2 O2
treatment of contaminated lettuce on transcription of acid stress
genes (gadA, gadB, and gadW), cold shock (cspA, cspC, and cspE),
and gene related to UV radiation stress (mutS) (Figures 1–6).
A fold change cutoff of 1.5 was applied in this study.
In E. coli O157:H7 present on lettuce, the genes associated
with oxidative stress (oxyR and sodA), universal stress (uspA
and rpoS), starvation (phoA), acid stress (gadB and gadW), and
virulence (stx1A, stx2A, and fliC) were significantly (P ≤ 0.05)
downregulated after exposure to 50 mM H2 O2 . The gadA gene
was downregulated 1.9-fold and the expression of soxR was below
the detectable level. Interestingly, two genes related to cold shock
(cspC and cspE) were significantly (P ≤ 0.05) upregulated. Genes
related to starvation (dps), cold shock (cspA), and mismatch
repair (MMR; mutS) were also upregulated (Figure 1).
In the case of E. coli O104:H4 on lettuce, H2 O2 treatment
caused significant (P ≤ 0.05) downregulation of genes related
to oxidative stress (oxyR and sodA), general stress (rpoS), acid
resistance (gadA, gadB, and gadW), and virulence (stx2A and
fliC). The uspA gene was also downregulated. Transcription of
dps, cspC, and mutS was significantly (P ≤ 0.05) upregulated.
In addition, gene encoding cold shock protein (cspE) was
upregulated (Figure 2).
In E. coli O145:NM present on lettuce, genes related to
oxidative damage (sodA), general stress (uspA and rpoS),
starvation (phoA), acid resistance (gadA and gadW), and
virulence (stx1A) were significantly (P ≤ 0.05) downregulated.
The soxR and gadB genes were also downregulated. Only two
genes (dps and cspE) were significantly (P ≤ 0.05) upregulated.
Furthermore, genes encoding cold shock protein (cspC), MMR
sensor (mutS), and intimin (eae) were upregulated (Figure 3).
In E. coli O111:NM on lettuce, nine genes, responsible
for oxidative damage (oxyR and sodA), general stress (uspA
and rpoS), starvation (phoA), acid resistance (gadA, gadB, and
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DISCUSSION
The effect of H2 O2 on the survival on lettuce of six VTEC
strains, representing O26:H11, O103:H2, O104:H4, O111:NM,
O145:NM, and O157:H7 serotypes, was investigated in this study.
A population reduction of 2.4–2.8 log10 was observed for all
VTEC strains on lettuce following treatment with 50 mM H2 O2
(Table 1). A previous study in our laboratory, using the same
VTEC strains suspended in TSB, reported greater sensitivity to
H2 O2 . For instance treatment, of pure broth culture of VTEC
O104:H4 with 1 mM H2 O2 resulted in a 2.7 log reduction and
2.5 mM H2 O2 caused a 3.7 log reduction in population of E. coli
O104:H4 (Tang and Kostrzynska, 2012). Therefore, the results
derived from these studies demonstrate that H2 O2 treatment
is less effective in lettuce decontamination compared to its
effect on reducing VTEC populations in pure cultures. Previous
investigations showed that several factors, such as organic loads
of fresh-cut produce (Gonzalez et al., 2004) , whole- or cut-leaf
wash (Nou and Luo, 2010), and leaf age (Brandl and Amundson,
2008), can influence the efficacy of sanitizer to inactivate E. coli
O157:H7 on fresh produce. These studies indicate that the lettuce
leaves may help to protect bacteria, making it harder to eliminate
pathogens from fresh-cut lettuce. Indeed, bacterial cells on lettuce
may be physically sequestered from H2 O2 exposure. In addition,
organic molecules released by fresh-cut lettuce will react with
H2 O2 and therefore reduce the effective exposure to H2 O2 .
Therefore, in order to choose an effective treatment to reduce
the pathogens on leafy greens, it is important to understand the
behavior of bacteria on lettuce under different stress conditions.
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FIGURE 1 | Effect of H2 O2 on gene transcription of E. coli O157:H7. Relative gene transcription represents the change in transcription compared to the
bacteria without H2 O2 treatment (control, value of 1.0). The transcription of each gene was normalized to the 16S rRNA transcription in each sample. Data are
expressed as the means ± SE for RNA extracted in three biological replicates. × denotes no transcription of soxR by E. coli O157:H7 after exposure to H2 O2 on
lettuce, however, soxR is present in this strain. ∗ denotes the significant change of gene transcription between treatment and control.
FIGURE 2 | Effect of H2 O2 on gene transcription of E. coli O104:H4. Relative gene transcription represents the change in transcription compared to the
bacteria without H2 O2 treatment (control, value of 1.0). The transcription of each gene was normalized to the 16S rRNA transcription in each sample. Data are
expressed as the means ± SE for RNA extracted in three biological replicates. × denotes absence of eae and stx1A genes in E. coli O104:H4. ∗ indicates the
significant change of gene transcription between treatment and control.
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FIGURE 3 | Effect of H2 O2 on gene transcription of E. coli O145:NM. Relative gene transcription represents the change in transcription compared to the
bacteria without H2 O2 treatment (control, value of 1.0). The transcription of each gene was normalized to the 16S rRNA transcription in each sample. Data are
expressed as the means ± SE for RNA extracted in three biological replicates. × denotes absence of stx2A and fliC genes in E. coli O145:NM. ∗ indicates the
significant change of gene transcription between treatment and control.
FIGURE 4 | Effect of H2 O2 on gene transcription of E. coli O111:NM. Relative gene transcription represents the change in transcription compared to the
bacteria without H2 O2 treatment (control, value of 1.0). The transcription of each gene was normalized to the 16S rRNA transcription in each sample. Data are
expressed as the means ± SE for RNA extracted in three biological replicates. × denotes absence of stx2A and fliC genes in E. coli O111:NM. ∗ indicates the
significant change of gene transcription between treatment and control.
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FIGURE 5 | Effect of H2 O2 on gene transcription of E. coli O26:H11. Relative gene transcription represents the change in transcription compared to the
bacteria without H2 O2 treatment (control, value of 1.0). The transcription of each gene was normalized to the 16S rRNA transcription in each sample. Data are
expressed as the means ± SE for RNA extracted in three biological replicates. × denotes absence of stx2A gene in E. coli O26:H11. ∗ indicates the significant
change of gene transcription between treatment and control.
FIGURE 6 | Effect of H2 O2 on gene transcription of E. coli O103:H2. Relative gene transcription represents the change in transcription compared to the
bacteria without H2 O2 treatment (control, value of 1.0). The transcription of each gene was normalized to the 16S rRNA transcription in each sample. Data are
expressed as the means ± SE for RNA extracted in three biological replicates. × denotes absence of stx2A gene in E. coli O103:H2. ∗ indicates the significant
change of gene transcription between treatment and control.
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of soxS and sodA. That study suggests that the expression of
oxyR or soxRS is dose-dependent. In addition, downregulation
of soxR was also observed in VTEC broth cultures exposed to
oxidative stress (Mei et al., 2015), however, the transcription of
sodA was significantly (P ≤ 0.05) increased in pure cultures of
all VTEC strains. The inconsistent results of expression of soxR
and sodA may indicate that (1) sodA can react to superoxide
stress independently; (2) SoxRS is not the sole regulator of sodA
gene.
To get a better understanding of the response to H2 O2
treatment in different VTEC serotypes, in the present study,
the transcription of genes related to stress and virulence in
O157:H7 and non-O157 serotypes on lettuce was evaluated.
Different gene transcription profiles following exposure to
H2 O2 were observed in VTEC strains on lettuce compared to
pure broth cultures (Mei et al., 2015). H2 O2 caused dramatic
downregulation of stress-associated and virulence genes in
VTEC strains present on lettuce, including O157 and non-157
serotypes. Ten genes were downregulated in all six VTEC
strains on lettuce (Figures 1–6). However, only three genes
were upregulated. These genes belong to different regulation
systems in E. coli and respond to various environmental
conditions by adjusting behavior accordingly to protect cells from
damaging.
General Stress Response (uspA and
rpoS) and Cold Shock Response (cspA,
cspC, and cspE)
Escherichia coli contains a large CspA family, consisting of
nine homologous cold inducible proteins, CspA to CspI, among
which, CspA is the major one produced at 10–24◦ C, and is
negatively downregulated by cspC. Whereas, CspC and CspE
are constitutively produced at 37◦ C and are not temperature
regulated (Yamanaka et al., 1994; Etchegaray et al., 1996; Phadtare
and Inouye, 2001). It has been reported that CspC and CspE
are important regulators of the expression of RpoS, a global
stress response regulator, and UspA, universal stress protein A
responding to various general stresses (Nystrom and Neidhardt,
1992, 1994). In addition, RpoS-controlled genes such as dps and
katG are upregulated or downregulated by the overexpression or
deletion of cspC and cspE (Phadtare and Inouye, 2001; Phadtare
et al., 2006). Therefore, CspC and CspE play important roles in
the stress response of E. coli. Upregulation of cspC and cspE in all
six VTEC strains on lettuce was observed in the present study
(Figures 1–6). However, uspA and rpoS were downregulated,
and a minor change in the transcription of dps was observed.
Interestingly, the transcription of cspC was downregulated in
broth cultures of all VTEC strains exposed to 2.5 mM H2 O2
at 37◦ C (Mei et al., 2015). On the other hand, in the present
study cspC was upregulated in VTEC strains on lettuce treated
with 50 mM H2 O2 at room temperature. Previous investigation
showed that CspC and CspE cannot be induced under some stress
conditions, like 0.5 M NaCl, 0.5 M KCl, 5% ethanol, pH 10,
pH 4, temperature of 15 or 50◦ C (Phadtare and Inouye, 2001).
It is possible that a different concentration of H2 O2 used for
lettuce decontamination as well as environmental factors such as
H2 O2 released from fresh-cut lettuce leaves as well as nutrients
present in lettuce samples and natural microbiota contributed
to the induction of cspC and cspE. More interestingly, contrary
results of transcription of cspC and cspE observed in response to
H2 O2 treatment of VTEC pure cultures and in the same strains
present on lettuce, suggest that VTEC may react to the same
stress differently in different environments. However, it is not
possible to rule out that induction of cold stress genes in present
study was more of the effect of temperature change combined
with peroxide treatment. Centrifugation of bacterial cells using
refrigerated centrifuge, following VTEC exposure on lettuce to
H2 O2 could influence transcription of cold shock genes. Further
studies are required to gain a better understanding of the stress
conditions that affect the cspC and cspE gene transcription in
VTEC present on lettuce.
Anti-Oxidant System Response (oxyR,
sodA, and soxR)
Superoxide dismutases (SODs) and catalases are employed by
E. coli to respond to superoxide and peroxide stress. There
are three SODs, including MnSOD (sodA), FeSOD (sodB),
and CuZnSOD (sodC), and two catalases (HPI and HPII,
encoded by katG and katE, respectively) in E. coli. These
SODs and catalase genes are regulated by two major oxidative
stress regulons, OxyR and SoxRS (Chiang and Schellhorn,
2012). In the presence of oxidative stress, OxyR can sense
H2 O2 and be converted to the oxidized form, subsequently
activating transcription of the OxyR regulon genes, such as
katG, dps, and oxyR, which protect the cell from H2 O2 toxicity
(Storz et al., 1990a; Farr and Kogoma, 1991; Chiang and
Schellhorn, 2012). In this study, the transcription of oxyR was
downregulated in all VTEC strains (Figures 1–6), this may
cause a reduction in catalase expression, increasing peroxide
sensitivity.
SoxRS is another key regulon triggered under oxidative stress
in E. coli. It is positively regulated by superoxide-generating
agents such as paraquat and constitutes a two-stage regulatory
system, in which SoxR, activated as a transcriptional activator,
induces the expression of soxS and the resulting increased
levels of SoxS protein regulate the transcription of the various
genes important for responding to oxidative stress (Demple
and Amabile-Cuevas, 1991; Nunoshiba et al., 1992; Chiang
and Schellhorn, 2012). The genes controlled by soxRS include
sodA (Mn-containing SOD), nfo (DNA repair endonuclease
IV), micF (antisense regulator of ompF), and zwf (glucose-6phosphate dehydrogenase) (Christman et al., 1985; Greenberg
et al., 1990; Tsaneva and Weiss, 1990). In the present study,
following exposure of VTEC strains on lettuce to 50 mM H2 O2 ,
the transcription of soxR in E. coli O103:H2 slightly increased
(1.5-fold), whereas, the transcription of soxR decreased to below
the detectable level in O157:H7. Therefore, SodA reduction
may be attributed to the reduction or only slight induction of
soxR. Manchado et al. (2000) reported that the expression of
OxyR-regulated genes (katG and dps) were induced when the
concentration of H2 O2 was from 1 to 100 µM, while the higher
concentration (≥500 µM H2 O2 ) resulted in the upregulation
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H2 O2 Treatment of Contaminated Lettuce
studies have shown that a total of 12 genes comprise an acid
fitness island, including a glutamate decarboxylase (GAD) system
and three transcriptional regulators (GadE, GadX, and GadW)
of the GAD enzymes, which renders E. coli the ability to survive
strong acidic stress (Hommais et al., 2001, 2004; Ma et al., 2002;
Bergholz et al., 2007; Mates et al., 2007). E. coli produce two
isozymes of GAD encoded by the gadA and gadB genes, which are
induced by GadX at any pH, while GadW represses expression
of gadX. GadW activates the expression of gadA and gadB only
in the absence of GadX (Ma et al., 2002; Tucker et al., 2003). In
addition, it has been reported that the expression of gadA and
gadBC were induced when bacteria were cultured in acidified
medium, treated with acetate, and during entry into stationary
phase (Selinger et al., 2000; Arnold et al., 2001; Tucker et al.,
2002). However, the expression of regulators GadX and GadW,
under the same conditions, were unknown. In the present study,
gadA, gadB, and gadW were significantly downregulated in all
six VTEC strains on lettuce (Figures 1–6). We suspect that the
expression of gadX was also downregulated in response to H2 O2 ,
which subsequently resulted in the downregulation of gadA and
gadB. Furthermore, the results indicate that following H2 O2
treatment, VTEC on lettuce may become more sensitive to acid
stress, which decreases the viability of pathogens under low pH.
Starvation Stress Response (phoA and
dps)
The gene phoA, encoding alkaline phosphatase, was induced
under a phosphate-limited condition, but was not synthesized
in normal growth medium (Han and Lee, 2006). The phoA is
a member of pho regulon, regulated by a two-protein system
PhoR–PhoP (Sola-Landa et al., 2003). The phoA was significantly
downregulated in all the VTEC strains on lettuce tested in this
study (Figures 1–6). Furthermore, downregulation of phoA was
observed in most VTEC strains when pure cultures were treated
with H2 O2 (Mei et al., 2015). Therefore, further studies are
needed to test, if H2 O2 treatment changes the sensitivity of VTEC
strains to phosphate-limited condition.
The H2 O2 can cause lethality of the bacterial cells through
several mechanisms. It has been proposed that the primary
cause of cell inactivation by H2 O2 or other oxidative agents is
DNA damage (Storz et al., 1990b). Thus, it is important for
the cells to cope with stresses by inducing the production of a
variety of DNA repair enzymes as well as catalases. Glutathione
also acts to protect cells from oxidative stress. In addition,
Dps—the DNA binding protein from starved cells, protects cells
during environmental stresses, including oxidative stress and
nutritional deprivation (Almiron et al., 1992; Calhoun and Kwon,
2011). Dps protects cells from harmful oxidative radicals by
DNA binding, iron storage, and by binding and oxidizing Fe
ions at ferroxidase centers. Furthermore, Dps may regulate the
expression of DNA repair enzymes and catalases necessary for
stress resistance. Zheng et al. (2001) reported that the expression
of dps considerably increased when the cells were exposed to
1 mM H2 O2 for 10 min. However, only minor upregulation of
dps was observed in this study for most of VTEC strains on lettuce
(Figures 1–6).
Virulence Factors (Encoded by eae,
stx1A, stx2A, and fliC Genes)
Production of Stx is the definitive virulence factor of VTEC
O157:H7 and non-O157 serotypes (Croxen et al., 2013; Scheutz,
2014; Smith et al., 2014; Franz et al., 2015). Stx produced by VTEC
can be classified into two types Stx1 and Stx2. There are three
subtypes of Stx1 (a, c, d) and seven subtypes of Stx2 (a, b, c, d, e, f,
g). Although both toxins could cause bloody diarrhea and HUS,
a specific subset of stx2 subtypes (stx2a, stx2c, and stx2d) have a
higher association with HC and HUS than stx1 subtypes or other
stx2 subtypes (Scheutz, 2014). Stx are encoded on bacteriophage
genomes that are integrated into bacterial chromosome. As such,
the biology of the Stx-encoding phages influences the expression
of stx1 and stx2. Production and release of toxins depend on
induction of these bacteriophages (Croxen et al., 2013; Scheutz,
2014). Amongst the VTEC strains tested in this study, E. coli
O157:H7 produced both Stx1 and Stx2, E. coli O104:H4 produced
only Stx2, and the remaining four strains produced only Stx1.
In this study, both stx1A and stx2A genes were dramatically
downregulated in all VTEC strains on lettuce following exposure
to H2 O2 (Figures 1–6).
Many VTEC strains produce attaching and effacing (AE)
lesions, which are controlled by the locus of enterocyte
effacement (LEE; Croxen et al., 2013; Scheutz, 2014). Although
AE lesions are not essential for bloody diarrhea and HUS, the
majority of strains implicated in these syndromes are LEEpositive. Intimin, encoded by eae (E. coli attaching and effacing)
gene, is responsible for intimate adhesion of VTEC to the
intestinal epithelium and formation of AE lesions. Intimin binds
to the cell receptor Tir, which is translocated by the pathogen
to the enterocyte via a type III secretion system (Croxen et al.,
2013). The presence of eae gene is strongly correlated to the
Mismatch Repair Response (mutS)
MutS is a member of methylation-dependent MMR system which
helps to maintain chromosome stability in E. coli. MutS binds to
the mismatches and initiates the long-patch MMR on daughter
DNA strands (Modrich and Lahue, 1996; Schofield and Hsieh,
2003). MMR has also been shown to be involved in the repair
of oxidative DNA damage which causes spontaneous lesion 7,8dihydro-8-oxo-guanine (8-oxoG or GO; Michaels and Miller,
1992; Tchou and Grollman, 1993; Lu et al., 2001). Studies have
shown that the amount of MutS in E. coli remarkably decreased
when cells were in the stationary phase and under starvation
stress and the expression of MutS repair protein was negatively
regulated by the RpoS and Hfq global regulators (Feng et al.,
1996; Tsui et al., 1997; Li et al., 2003). In the present study, mutS
was upregulated following exposure of VTEC on lettuce to H2 O2
(Figures 1–6). This upregulation may be caused by significantly
downregulated expression of RpoS, which is a negative regulator
of mutS.
Acid Response (gadA, gadB, and gadW)
Food-borne pathogenic E. coli must be able to survive in the
extremely acidic environment of the stomach and resist very low
pH (1.5–3.0) for several hours (Peterson et al., 1989). Previous
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H2 O2 Treatment of Contaminated Lettuce
VTEC serotypes. Consequently, VTEC strains on lettuce may
become more sensitive to acid stress following H2 O2 treatment.
Interestingly, different gene transcription patterns in response
to H2 O2 were observed in VTEC strains on lettuce compared
to pure broth cultures (Mei et al., 2015). Particularly, sodA
gene encoding manganese SOD was significantly upregulated
in pure cultures of all VTEC serotypes, however, this gene
was downregulated in the same strains present on lettuce. In
addition, Stx genes were upregulated in broth culture of E. coli
O157:H7 and downregulated in most VTEC serotypes (including
O157:H7) on lettuce following H2 O2 treatment. It is possible
that factors released from fresh-cut lettuce leaves and natural
microbiota present in lettuce samples contributed to different
gene transcription patterns in VTEC strains on lettuce compared
to broth cultures.
presence of genes encoding many other virulence factors (Franz
et al., 2015). In addition, VTEC strains with eae and stx2 genes
have been associated with HUS and bloody diarrhea (Scheutz,
2014). In the present study, the transcription of eae (except for
E. coli O104:H4 which is LEE-negative) changed only slightly
following H2 O2 treatment (Figures 1–6). Thus, further studies
about eae regulation and the intimin-related virulence of VTEC
strains under different stress conditions are needed.
Production of flagella is another contributor to the
pathogenicity of VTEC (Gyles, 2007). Flagella are mainly
responsible for motility, chemotaxis, and secretion of virulence
factors. The key structural component of the flagellum filament is
encoded by the fliC gene. In our studies, the transcription of fliC
was dramatically downregulated in all four motile VTEC strains
on lettuce treated with H2 O2 (Figures 1–6). In addition, fliC
was downregulated in pure broth cultures exposed to H2 O2 (Mei
et al., 2015). These results suggest that H2 O2 treatment affects
motility of VTEC strains.
AUTHOR CONTRIBUTIONS
All authors have made substantial direct and intellectual
contributions to this work.
CONCLUSION
H2 O2 treatment caused less dramatic reduction in VTEC
populations on lettuce compared to pure broth cultures, which
indicates that VTEC are protected from H2 O2 on leafy greens.
Consequently higher concentration of H2 O2 or other sanitizers
are required to reduce and eliminate VTEC on lettuce. As such,
the results derived from this study showed the importance of
food matrix in studying the effect of sanitizers on survival
rate of VTEC. In addition, VTEC strains on lettuce showed
similar transcription patterns (regardless of serotype) in response
to treatment with 50 mM H2 O2. The transcription of genes
related to oxidative stress, general stress, acid stress and
some virulence genes were significantly downregulated in six
FUNDING
This research was supported by the Agriculture and Agri-Food
Canada.
ACKNOWLEDGMENTS
Authors would like to thank Dr. Roger Johnson of Public Health
Agency of Canada and Dr. Pascal Delaquis (AAFC) for non-O157
VTEC strains.
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March 2017 | Volume 8 | Article 477